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    • A nuance of the conventional conditions has been that while

    2018-10-24

    A nuance of the conventional conditions has been that while overall amplification of cell number occurs in the cultures, the percentage of the population possessing regenerative capacity (i.e., SSCs) declines over a several-month period (Kanatsu-Shinohara et al., 2003, 2005, 2011; Kubota et al., 2004b; Schmidt et al., 2011), a deficiency that is amplified when the germ cells are maintained in a feeder-free format (Kanatsu-Shinohara et al., 2011). There are several possible causes of the declining SSC content, including dilution of the pool by greater proliferation of the non-stem cell progenitor spermatogonia or impaired integrity of SSCs to self-renew. At present, experimental evidence supporting a singular or multifactor deficiency has not been generated, and further experimentation will be required to clarify the cause of reduced maintenance of the SSC pool. Also, the integrity of the colony-producing population for regenerating complete spermatogenesis at various points during the culture period has not been defined. In an effort to enhance growth of the cultures overall and improve maintenance of the SSC pool, Kanatsu-Shinohara et al. (2011) refined the StemPro-based culture medium to be serum free and lipid enriched. In those conditions, the growth of cultures overall was improved and a pool of cells with regenerative capacity persisted for an extended period of time. In follow-up studies, the same group aimed to improve the serum-free and lipid-rich condition by changing the commercially available PCP inhibitor medium (Kanatsu-Shinohara et al., 2014). Outcomes yielded a condition whereby the percentage of the culture population that is capable of regenerating spermatogenesis was maintained at a relatively steady level over a 5-month period. However, for both of these refined conditions, assessment of the percentage of the population with prime stem cell capacity to produce colonies of complete spermatogenesis after transplant at various points during the culture period was not conducted. Also, similar to the unrefined conditions, the cultures were derived from prospermatogonia of P0 neonates with a DBA/2 genetic background; thus, relevance to the true undifferentiated spermatogonial population and other strains of mice has remained undefined. For many somatic cell lineages, the bioenergetics of stem cells is unique compared with progenitors and differentiated cells, with most relying on glycolysis (Hsu et al., 2013; Ito and Suda, 2014; Simsek et al., 2010). If this is the case for SSCs, modifying conditions to favor glycolysis could be of benefit for maintaining regenerative integrity of the pool during long-term culture. Indeed, our previous studies that examined the transcriptome of the SSC pool in primary cultures of undifferentiated spermatogonia derived from P6–P8 mice with a C57BL/6 genetic background revealed differential expression of key enzymes (Eno1/2) that are involved in glycolysis (Chan et al., 2014). Expanding on this observation, a recent study found that inhibition of glycolysis in prospermatogonia-derived cultures derived from DBA/2 mice reduced the number of cells with regenerative capacity, whereas chemical activation of glycolysis enhanced stem cell maintenance (Kanatsu-Shinohara et al., 2016). Paradoxically, because an abundance of free fatty acids will promote utilization of β-oxidation, the findings that modulating glycolytic activity improves SSC self-renewal contradicts the previous studies suggesting that a lipid-rich medium enhances SSC maintenance during long-term culture (Kanatsu-Shinohara et al., 2011, 2014). Unfortunately, the SSC content was not reported at various points during an extended culture period in the study by Kanatsu-Shinohara et al. (2016), so the impact of modulating glycolytic activity on long-term maintenance of the SSC pool is not clear. Interestingly, by adding a chemical activator of glycolysis to culture medium, Kanatsu-Shinohara et al. (2016) were able to derive primary cultures from mice with a C57BL/6 genetic background, a feat they had not been able to achieve using the StemPro and lipid-rich condition that works efficiently for cells from DBA/2 mice. Although the percentage of the cultured populations that possessed regenerative capacity was reported to be similar between C57BL/6 and DBA/2 cultures when maintained with supplementation of the glycolysis activator, the length of time that the cells had been maintained in vitro prior to transplant was not reported. In addition, the percentage of colonies that formed in recipient testes after transplantation that were complete spermatogenesis was not assessed. Thus, the effects of modulating the bioenergetics in primary cultures of undifferentiated spermatogonia on long-term maintenance of SSC regenerative integrity have remained undefined.